Nutritional Factors Modulating Alu Methylation inan Italian Sample from The Mark-Age StudyIncluding Offspring of Healthy Nonagenarians

Alu hypomethylation promotes genomic instability and is associated with aging and age-related diseases. Dietary factors affect global DNA methylation, leading to changes in genomic stability and gene expression with an impact on longevity and the risk of disease. This preliminary study aims to investigate the relationship between nutritional factors, such as circulating trace elements, lipids and antioxidants, and Alu methylation in elderly subjects and offspring of healthy nonagenarians. Alu DNA methylation was analyzed in sixty RASIG (randomly recruited age-stratified individuals from the general population) and thirty-two GO (GeHA offspring) enrolled in Italy in the framework of the MARK-AGE project. Factor analysis revealed a different clustering between Alu CpG1 and the other CpG sites. RASIG over 65 years showed lower Alu CpG1 methylation than those of GO subjects in the same age class. Moreover, Alu CpG1 methylation was associated with fruit and whole-grain bread consumption, LDL2-Cholesterol and plasma copper. The preserved Alu methylation status in GO, suggests Alu epigenetic changes as a potential marker of aging. Our preliminary investigation shows that Alu methylation may be affected by food rich in fibers and antioxidants, or circulating LDL subfractions and plasma copper

Obesity accelerates epigenetic aging in middle-aged but not in elderly individuals

Background: Human aging is associated with profound changes in one of the major epigenetic mechanisms, DNA methylation. Some of these changes occur in a clock-like fashion, i.e., correlating with the calendar age of an individual, thus providing a new aging biomarker. Some reports have identified factors associated with the acceleration of the epigenetic age. However, it is also important to analyze the temporal changes in the epigenetic age, i.e., the duration of the observed acceleration, and the effects of the possible therapeutic and lifestyle modifications.Methods: To address this issue, we determined the epigenetic age for a cohort of 183 healthy individuals using blood samples derived from two time points that were 25 years apart (between 15-24 and 40-49 years of age). Additionally, we also determined the epigenetic ages of 119 individuals in a cohort consisting of 90-year-old participants (nonagenarians). These were determined by using the Horvath algorithm based on the methylation level of 353 CpG sites. The data are indicated as the deviation of the epigenetic age from the calendar age (calendar age minus epigenetic age = delta age, Delta AGE). As obesity is often associated with accelerating aging and degenerative phenotypes, the correlation of the body mass index (BMI) with the Delta AGE was analyzed in the following three age groups: young adults, middle-aged, and nonagenarian.Results: The data showed that BMI is associated with decreased Delta AGE, i.e., increased epigenetic age, in middle-aged individuals. This effect is also seen during the 25-year period from early adulthood to middle age, in which an increase in the BMI is significantly associated with a decrease in the Delta AGE. We also analyzed the association between BMI and epigenetic age in young and elderly individuals, but these associations were not significant.Conclusion: Taken together, the main finding on this report suggests that association between increased BMI and accelerated epigenetic aging in the blood cells of middle-aged individuals can be observed, and this effect is also detectable if the BMI has increased in adulthood. The fact that the association between BMI and epigenetic age can only be observed in the middle-aged group does not exclude the possibility that this association could be present throughout the human lifespan; it might just be masked by confounding factors in young adults and nonagenarian individuals

Association between fat-soluble vitamins and self-reported health status: A cross-sectional analysis of the MARK-AGE cohort

Self-rated health (SRH) is associated with higher risk of death. Since low plasma levels of fat-soluble vitamins are related to mortality, we aimed to assess whether plasma concentrations of vitamins A, D and E were associated with SRH in the MARK-AGE study. We included 3158 participants (52% female) aged between 35-75 years. Cross-sectional data were collected via questionnaires. An enzyme immunoassay quantified 25-hydroxyvitamin D and HPLC determined α-tocopherol and retinol plasma concentrations. The median 25-hydroxyvitamin D and retinol concentrations differed significantly (P<0.001) between SRH categories, and were lower in the combined fair/poor category versus the excellent, very good, good categories (25-hydroxvitamin D: 40.8 vs. 51.9, 49.3, 46.7 nmol/l, respectively; retinol: 1.67 vs. 1.75, 1.74, 1.70 μmol/l, respectively). Both vitamin D and retinol status were independently associated with fair/poor SRH in multiple regression analyses: adjusted ORs (95% CI) for the vitamin D insufficiency, deficiency, severe deficiency categories were 1.33 (1.06-1.68), 1.50 (1.17-1.93), and 1.83 (1.34-2.50) respectively; P=0.015, P=0.001, P<0.001, and for the second/third/fourth retinol quartiles: 1.44 (1.18-1.75), 1.57 (1.28-1.93), 1.49 (1.20-1.84); all P<0.001. No significant associations were reported for α-tocopherol quartiles. Lower vitamin A and D status emerged as independent markers for fair/poor SRH. Further insights into the long-term implications of these modifiable nutrients on health status are warranted

Bacterial DNAemia in Older Participants and Nonagenarian Offspring and Association With Redox Biomarkers: Results From MARK-AGE Study

Aging and age-related diseases have been linked to microbial dysbiosis with changes in blood bacterial DNA concentration. This condition may promote chronic low-grade inflammation, which can be further aggravated by antioxidant nutrient deficiency. Low plasma carotenoids are associated with an increased risk of inflammation and cellular damage and predict mortality. However, no evidence is yet available on the relationship between antioxidants and the blood bacterial DNA (BB-DNA). Therefore, this study aimed to compare BB-DNA from (a) GO (nonagenarian offspring), (b) age-matched controls (Randomly recruited Age-Stratified Individuals from the General population [RASIG]), and (c) spouses of GO (SGO) recruited in the MARK-AGE project, as well as to investigate the association between BB-DNA, behavior habits, Charlson Comorbidity Index (CCI), leucocyte subsets, and the circulating levels of some antioxidants and oxidative stress markers. BB-DNA was higher in RASIG than GO and SGO, whereas GO and SGO participants showed similar values. BB-DNA increased in smokers and males with CCI >= 2 compared with those with CCI <= 1 within RASIG. Moreover, BB-DNA was positively associated with lymphocyte, neutrophil, and monocyte counts, but not with self-reported dietary habits. Higher quartiles of BB-DNA were associated with low lutein and zeaxanthin and elevated malondialdehyde plasma concentrations in RASIG. BB-DNA was also positively correlated with nitric oxide levels. Herein, we provide evidence of a reduced BB-DNA in individuals from long-living families and their spouses, suggesting a decreased microbial dysbiosis and bacterial systemic translocation. BB-DNA was also associated with smoking, CCI, leukocyte subsets, and some redox biomarkers in older participants

Somatic Point Mutations in mtDNA Control Region Are Influenced by Genetic Background and Associated with Healthy Aging: A GEHA Study

Tissue specific somatic mutations occurring in the mtDNA control region have been proposed to provide a survival advantage. Data on twins and on relatives of long-lived subjects suggested that the occurrence/accumulation of these mutations may be genetically influenced. To further investigate control region somatic heteroplasmy in the elderly, we analyzed the segment surrounding the nt 150 position (previously reported as specific of Leukocytes) in various types of leukocytes obtained from 195 ultra-nonagenarians sib-pairs of Italian or Finnish origin collected in the frame of the GEHA Project. We found a significant correlation of the mtDNA control region heteroplasmy between sibs, confirming a genetic influence on this phenomenon. Furthermore, many subjects showed heteroplasmy due to mutations different from the C150T transition. In these cases heteroplasmy was correlated within sibpairs in Finnish and northern Italian samples, but not in southern Italians. This suggested that the genetic contribution to control region mutations may be population specific. Finally, we observed a possible correlation between heteroplasmy and Hand Grip strength, one of the best markers of physical performance and of mortality risk in the elderly. Our study provides new evidence on the relevance of mtDNA somatic mutations in aging and longevity and confirms that the occurrence of specific point mutations in the mtDNA control region may represent a strategy for the age-related remodelling of organismal functions

Association of Torquetenovirus Viremia with Physical Frailty and Cognitive Impairment in Three Independent European Cohorts

Introduction: Immunosenescence and inflammaging have been implicated in the pathophysiology of frailty. Torquetenovirus (TTV), a single-stranded DNA anellovirus, the major component of the human blood virome, shows an increased replication rate with advancing age. An elevated TTV viremia has been associated with an impaired immune function and an increased risk of mortality in the older population. The objective of this study was to analyze the relation between TTV viremia, physical frailty, and cognitive impairment. Methods: TTV viremia was measured in 1,131 nonfrail, 45 physically frail, and 113 cognitively impaired older adults recruited in the MARK-AGE study (overall mean age 64.7 ± 5.9 years), and then the results were checked in two other independent cohorts from Spain and Portugal, including 126 frail, 252 prefrail, and 141 nonfrail individuals (overall mean age: 77.5 ± 8.3 years). Results: TTV viremia ≥4log was associated with physical frailty (OR: 4.69; 95% CI: 2.06-10.67, p < 0.0001) and cognitive impairment (OR: 3.49, 95% CI: 2.14-5.69, p < 0.0001) in the MARK-AGE population. The association between TTV DNA load and frailty status was confirmed in the Spanish cohort, while a slight association with cognitive impairment was observed (OR: 1.33; 95% CI: 1.000-1.773), only in the unadjusted model. No association between TTV load and frailty or cognitive impairment was found in the Portuguese sample, although a negative association between TTV viremia and MMSE score was observed in Spanish and Portuguese females. Conclusions: These findings demonstrate an association between TTV viremia and physical frailty, while the association with cognitive impairment was observed only in the younger population from the MARK-AGE study. Further research is necessary to clarify TTV's clinical relevance in the onset and progression of frailty and cognitive decline in older individuals

Differentiation in Neuroblastoma: Diffusion-Limited Hypoxia Induces Neuro-Endocrine Secretory Protein 55 and Other Markers of a Chromaffin Phenotype

Background: Neuroblastoma is a childhood malignancy of sympathetic embryonal origin. A high potential for differentiation is a hallmark of neuroblastoma cells. We have previously presented data to suggest that in situ differentiation in tumors frequently proceeds along the chromaffin lineage and that decreased oxygen ( hypoxia) plays a role in this. Here we explore the utility of Neuro-Endocrine Secretory Protein 55 ( NESP55), a novel member of the chromogranin family, as a marker for this process.Methodology/Principal Findings: Immunohistochemical analyses and in situ hybridizations were performed on human fetal tissues, mouse xenografts of human neuroblastoma cell lines, and on specimens of human neuroblastoma/ganglioneuroma. Effects of anaerobic exposure on gene expression by cultured neuroblastoma cells was analyzed with quantitative real-time PCR. Fetal sympathetic nervous system expression of NESP55 was shown to be specific for chromaffin cell types. In experimental and clinical neuroblastoma NESP55 immunoreactivity was specific for regions of chronic hypoxia. NESP55 expression also correlated strikingly with morphological evidence of differentiation and with other chromaffin-specific patterns of gene expression, including IGF2 and HIF2 alpha. Anaerobic culture of five neuroblastoma cell lines resulted in an 18.9-fold mean up-regulation of NESP55.Conclusions/Significance: The data confirms that chronic tumor hypoxia is a key microenvironmental factor for neuroblastoma cell differentiation, causing induction of chromaffin features and NESP55 provides a reliable marker for this neuronal to neuroendocrine transition. The hypoxia-induced phenotype is the predominant form of differentiation in stroma-poor tumors, while in stroma-rich tumors the chromaffin phenotype coexists with ganglion cell-like differentiation. The findings provide new insights into the biological diversity which is a striking feature of this group of tumors

The bank lending channel: an empirical assessment of measures to stimulate bank lending in the European Union

This thesis first examines the role of banks in the transmission mechanism of monetary policy by focusing on the eight European new member States of Central and Eastern Europe over the 2004-2013 period. We specifically investigate the influence of monetary policy changes on bank lending activity and if this potential influence is contingent on bank characteristics, such as banks’ size, capital, liquidity, risk factor and market power. Moreover, we focus on the prospective role of banks in the monetary policy transmission mechanism in order to reveal any clear trends in banks’ lending behaviour during the 2008-2011 financial crisis. Secondly, we investigate the impact of a protracted period of low monetary policy rates on loosening of banks’ credit standards regarding enterprises, households and consumer loans through concentrating on the nine Eurozone countries involved since the initiation of the Euro area Bank Lending Survey in the three distinct time frames of pre- (2002Q4-2008Q3), mid- (2008Q4-2010Q4) and post- (2011Q1-2014:Q4) financial crisis. Furthermore, we test the fundamental concept of the risk taking channel by examining excessive risk-taking behaviour by banks in stressed vs. non-stressed countries of the Eurozone. In an additional analysis, the efficacy of the European Central Bank’s 3 year Long-Term Refinancing Operations is evaluated in great depth in order to determine whether banks’ credit standards have been softened and the degree to which demand for loans has increased. Thirdly, we explore the financing structure of bank lending constrained Small and Medium Sized Enterprises in the eleven Eurozone countries by utilising firm-level data over the period of 2009 to 2014. We estimate if bank lending constrained firms demonstrate relatively more usage or requests for alternative financing. Additionally, a comprehensive investigation is presented by unveiling the impact and determinants of various financing constraints including credit lines, bank loans, trade credit and other lending on Eurozone firms. Furthermore, the notion of discouraged borrowers originally formulated by Kon and Storey (2003) is empirically evaluated. Finally, we present the conclusion of our research by further outlining its limitations and prospective scope for future studie

In Vivo Methods to Study Uptake of Nanoparticles into the Brain

Several in vivo techniques have been developed to study and measure the uptake of CNS compounds into the brain. With these techniques, various parameters can be determined after drug administration, including the blood-to-brain influx constant (Kin), the permeability-surface area (PS) product, and the brain uptake index (BUI). These techniques have been mostly used for drugs that are expected to enter the brain via transmembrane diffusion or by carrier-mediated transcytosis. Drugs that have limitations in entering the brain via such pathways have been encapsulated in nanoparticles (based on lipids or synthetic polymers) to enhance brain uptake. Nanoparticles are different from CNS compounds in size, composition and uptake mechanisms. This has led to different methods and approaches to study brain uptake in vivo. Here we discuss the techniques generally used to measure nanoparticle uptake in addition to the techniques used for CNS compounds. Techniques include visualization methods, behavioral tests, and quantitative methods

Ageing affects subtelomeric DNA methylation in blood cells from a large European population enrolled in the MARK-AGE study

Ageing leaves characteristic traces in the DNA methylation make-up of the genome. However, the importance of DNA methylation in ageing remains unclear. The study of subtelomeric regions could give promising insights into this issue. Previously reported associations between susceptibility to age-related diseases and epigenetic instability at subtelomeres suggest that the DNA methylation profile of subtelomeres undergoes remodelling during ageing. In the present work, this hypothesis has been tested in the context of the European large-scale project MARK-AGE. In this cross-sectional study, we profiled the DNA methylation of chromosomes 5 and 21 subtelomeres, in more than 2000 age-stratified women and men recruited in eight European countries. The study included individuals from the general population as well as the offspring of nonagenarians and Down syndrome subjects, who served as putative models of delayed and accelerated ageing, respectively. Significant linear changes of subtelomeric DNA methylation with increasing age were detected in the general population, indicating that subtelomeric DNA methylation changes are typical signs of ageing. Data also show that, compared to the general population, the dynamics of age-related DNA methylation changes are attenuated in the offspring of centenarian, while they accelerate in Down syndrome individuals. This result suggests that subtelomeric DNA methylation changes reflect the rate of ageing progression. We next attempted to trace the age-related changes of subtelomeric methylation back to the influence of diverse variables associated with methylation variations in the population, including demographics, dietary/health habits and clinical parameters. Results indicate that the effects of age on subtelomeric DNA methylation are mostly independent of all other variables evaluated.Molecular Epidemiolog